Assays and methods for cell proliferation-targeted treatment therapies

Abstract

Assays are described which measure polyamine transport activity and markers associated with polyamine transport and metabolism. These data are then used for the selection of treatments using therapies which target polyamine transport and polyamine metabolism. The assay includes a substrate to which a protein-containing cell sample can bind, a solution comprising a first antibody specific against ATP13A3, wherein the first antibody is configured to bind to ATP13A3 protein on the substrate, a solution comprising a second antibody specific against the first antibody, the second antibody comprising an enzyme linked thereto, wherein the second antibody is configured to bind to the first antibody. The assay further includes a substrate specific to the enzyme, wherein upon combining the substrate and the enzyme, the amount of enzyme in the solution can be identified, wherein said amount of enzyme identified is proportional to the amount of ATP13A3 protein in the sample, wherein said ATP13A3 protein is indicative of polyamine transport in the cells of the sample. In addition, primary antibodies covalently attached to respective fluorophores can be used to directly measure the relative expression levels of these biomarkers in histological samples. In addition, several biomarkers are described which allow for therapy selection based upon the expression and relative ratios of specific proteins associated with polyamine transport (c-myc, ATP13A3 Cav-2, and Cav-1 as well as c-Raf).

What is claimed is: 
     
         1 . A method for detecting cancer cells susceptible to treatment with a polyamine transport inhibitor (PTI), comprising:
 receiving a cell sample from a patient;   
       presenting a first reagent, a second reagent, a third reagent, and a fourth reagent to the cell sample to detect differential levels of ATP13A3, c-myc, Cav-1, and Cav-2 or nucleic acid sequences encoding the same, in the cell sample, wherein the first reagent is specific to ATP13A3, the second reagent is specific to c-myc, the third reagent is specific to Cav-1, and the fourth reagent is specific to Cav-2 in the cell sample, under conditions to detect binding of the first, second, third and fourth reagents to ATP13A3, c-myc, Cav-1, and Cav-2, respectively, or hybridization of the first, second, third and fourth reagents to the nucleic acid sequences,
 determining that cancer cells in the patient are treatable with a polyamine transport inhibitor if a higher level of ATP13A3, Cav-2, and c-myc and a basal or low level of Cav-1, or nucleic acid sequences encoding same, than in normal cells was determined to be present in the cell sample. 
 
     
     
         2 . The method of  claim 1  wherein first, second, third and fourth reagents are antibodies. 
     
     
         3 . The method of  claim 2 , wherein conditions to detect binding of the first, second, third and fourth reagents comprises conducting an enzyme linked immunosorbent assay or a western blot method, wherein the first, second, third and fourth reagents comprise a detectable marker. 
     
     
         4 . The method of  claim 1 , wherein the first reagent, second reagent, third reagent, and fourth reagent are primers directed to sequences that encode ATP13A3, c-myc, Cav-1, and Cav-2 respectively, and wherein conditions to detect hybridization comprises subjecting the cell sample and first, second, third and fourth reagents to PCR. 
     
     
         5 . The method of  claim 1 , wherein the cell sample comprises intact cells or tissue, and the first reagent comprises a first antibody A  to detect ATP13A3, the second reagent comprises a first antibody B  to detect c-myc, the third reagent comprises a first antibody C  to detect Cav-1, and the fourth reagent comprises a first antibody D  to detect Cav-2 in the cell sample, and wherein conditions to detect binding comprises conducting an immunocytochemistry or immunohistochemistry method on the cell sample. 
     
     
         6 . The method of  claim 5 , wherein the first antibodies each comprise a label. 
     
     
         7 . The method of  claim 5 , further comprising presenting a second antibody A  specific to and capable of binding the first antibody A , a second antibody B  specific to and capable of binding the first antibody B , a second antibody C  specific to and capable of binding the first antibody C , and a second antibody D  specific to and capable of binding the first antibody D . 
     
     
         8 . The method of  claim 7 , wherein each of the second antibodies comprise a label. 
     
     
         9 . (canceled) 
     
     
         10 . (canceled) 
     
     
         11 . (canceled) 
     
     
         12 . The method of  claim 1 , wherein the method further comprises identifying high levels of expression of c-Raf in the cancer cells. 
     
     
         13 . The method of  claim 1 , further comprising administering a therapeutically effective amount of a polyamine biosynthesis inhibitor and/or a PTI to the patient upon determining cancer cells in the patient are treatable. 
     
     
         14 - 69 . (canceled) 
     
     
         70 . An assay system to identify cells having polyamine transport activity (PTA) for targeted treatment of cells, said assay system comprising:
 a substrate configured to bind proteins in a cell sample; and   two or more antibodies selected from the group consisting of a primary antibody A  that detects ATP13A3, a primary antibody B  that detects c-myc, a primary antibody C  that detects Cav-1 and a primary antibody D  that detects Cav-2 in a sample, wherein upon contacting the cell sample and the primary antibodies A-D  to the substrate, the primary antibodies A-D  bind specifically to proteins ATP13A3, c-myc, Cav-1, and Cav-2, respectively, in the cell sample, wherein the primary antibodies A-D  are each individually linked to different colored fluorophores, such that visualization comprises a colored panel readout of the relative expression of each protein and differential expression levels of the proteins can be detected,   and wherein a detection of high levels of ATP13A3, c-myc, and/or Cav-2, and/or a low level of Cav-1 in the sample is indicative of PTA (polyamine transport activity) in the cell sample.   
     
     
         71 . (canceled) 
     
     
         72 . (canceled) 
     
     
         73 . A method for detecting cancer cells susceptible to treatment with a polyamine transport inhibitor (PTI), comprising:
 receiving a cell sample from a patient;   presenting at least a first and a second reagent to the cell sample to detect differential levels of two or more of ATP13A3, c-myc, Cav-1, and Cav-2 or nucleic acid sequences encoding the same, in the cell sample, under conditions to detect binding of the at least first and second reagents to two or more of ATP13A3, c-myc, Cav-1, and Cav-2, or hybridization of at least the first and second reagents to nucleic acid sequences.   
     
     
         74 . The method of  claim 73 , wherein the first and second reagents are specific to ATP13A3 and Cav-1, respectively, and wherein the method further comprises determining that cancer cells in the patient are treatable with a polyamine transport inhibitor if a higher level of ATP13A3 and a lower or basal level of Cav-1, or nucleic acid sequences encoding same, relative to normal cells, was determined to be present in the cell sample; and wherein the hi her level of ATP13A3 is at least 50-100% higher than basal level, and wherein the lower level of Cav-1 is within 20% of basal level, relative to normal cells. 
     
     
         75 . (canceled) 
     
     
         76 . The method of  claim 73 , wherein the first and second reagents are specific to ATP13A3 and c-myc, respectively, and wherein the method further comprises determining that cancer cells in the patient are treatable with a polyamine transport inhibitor if a higher level of ATP13A3 and a higher level of c-myc, or nucleic acid sequences encoding same, relative to normal cells was determined to be present in the cell sample; and wherein the higher level of ATP13A3 is at least 50-100% higher than basal level, and wherein higher level of c-myc is at least 50-100% higher than basal level, relative to normal cells. 
     
     
         77 . (canceled) 
     
     
         78 . The method of  claim 73 , wherein the first and second reagents are specific to Cav-1 and Cav-2, respectively, and wherein the method further comprises determining that cancer cells in the patient are treatable with a polyamine transport inhibitor if a higher level of Cav-2 and a lower or basal level of Cav-1, or nucleic acid sequences encoding same, relative to normal cells was determined to be present in the cell sample. 
     
     
         79 . The method of  claim 73 , wherein the first and second reagents are specific to ATP13A3 and Cav-2, respectively, and wherein the method further comprises determining that cancer cells in the patient are treatable with a polyamine transport inhibitor if a higher level of ATP13A3 and a higher level of Cav-2, or nucleic acid sequences encoding same, relative to normal cells was determined to be present in the cell sample; and wherein the higher level of ATP13A3 is at least 50-110% higher than basal level and wherein higher level of Cav-2 is within 20% of basal level relative to normal cells. 
     
     
         80 . (canceled) 
     
     
         81 . The method of  claim 73 , wherein the first and second reagents are specific to c-myc and Cav-1, respectively, and wherein the method further comprises determining that cancer cells in the patient are treatable with a polyamine transport inhibitor if a higher level of c-myc and a lower or basal level of Cav-1, or nucleic acid sequences encoding same, relative to normal cells was determined to be present in the cell sample, and wherein the higher level of c-myc is at least 50-100% higher than basal level and wherein the lower level of Cav-1 is within 20% of basal level. 
     
     
         82 . (canceled) 
     
     
         83 . The method of  claim 73 , wherein the first and second reagents are specific to c-myc and Cav-2, respectively, and wherein the method further comprises determining that cancer cells in the patient are treatable with a polyamine transport inhibitor if a higher level of c-myc and a higher level of Cav-2, or nucleic acid sequences encoding same, relative to normal cells was determined to be present in the cell sample; and wherein the higher level of c-myc is at least 50-100% higher than basal level, and wherein higher level of Cav-2 is at least 50-100% higher than basal level. 
     
     
         84 . (canceled) 
     
     
         85 . (canceled) 
     
     
         86 . (canceled) 
     
     
         87 . (canceled) 
     
     
         88 . (canceled) 
     
     
         89 . (canceled) 
     
     
         90 . The method of  claim 73 , wherein the first and second reagents are primary antibodies selected from a group consisting of a primary antibody A  that detects ATP13A3, a primary antibody B  that detects c-myc, a primary antibody C  that detects Cav-1, and a primary antibody D  that detects Cav-2 
     
     
         91 . The method of  claim 90 , wherein the primary antibody A  is HPA-029471.